Diarrhetic shellfish poisoning (DSP) is a gastrointestinal illness of varying severity caused by consumption of shellfish contaminated with certain dinoflagellates. It is caused by toxins which are accumulated in the midgut glands of bivalves feeding on dinoflagellates such as Dinophysis sp. and Prorocentrum sp. These types of organisms are part of the marine plankton shellfish ingest through filter feeding. Therefore, since considerable quantities of plankton are absorbed by filtering organisms, the bivalves may potentially accumulate substantial amounts of toxins.
During the period ranging from 1976 to 1982, over 1000 people were treated for gastroenteritis in Japan after having consumed boiled mussels, scallops and clams. Also, numerous cases of DSP have been reported in countries such as Mexico, Spain, The Netherlands, Scandinavia, France and South America. The high morbidity rate and worldwide distribution of DSPs make it a serious threat to both the shellfish industry and to public health.
One of the major components of the toxic profile associated with DSP is okadaic acid, although other polyether toxins such as yessotoxin, dinophysistoxin-3 and pectenotoxin may also be involved. Okadaic acid was first isolated from the marine sponges Halichondria okadaii and Halichondria melanodocia and reported by Tachibana et al. in 1981, J. Am. Chem. Soc. 103, 2469-2472.
In recent years, okadaic acid and dinophysistoxin-1 have been found to be powerful tumour promoters. In two-stage carcinogenesis experiments on mouse skin, these compounds were found to be of comparable potency to agents like phorbol esters. Because of their tumor promoting properties, currently acceptable quarantine levels for these toxins (200 ng/g shellfish tissue) therefore appear to be much higher than what should actually be tolerated. In fact, there is a strong possibility that, based on future injection studies in animals, shellfish contaminated even with minute amounts of DSP toxins will be declared unsuitable for human consumption. Hence, it would appear desirable to develop an assay which would permit the detection of very low concentrations of DSP toxins.
The analysis of DSP toxins such as okadaic acid and dinophysistoxin-1 presents a formidable challenge due to their low concentrations in complex mixtures and lack of a characteristic chromophore. The non-specific mouse bioassay has routinely been employed for monitoring DSP. However, there is considerable margin of error in its use and it is imprecise in defining a causative toxin in contaminated samples. This was discussed by Yasumoto et al. in 1985, Tetrahedron 41, 1019-1025.
Recently, a sensitive monoclonal antibody based assay, specific for okadaic acid-related DSP's, was developed as an easy to use field kit. This technology is described by Usagawa et al. in 1989, Toxicon 27, 1323-1330. A specific radioimmunoassay was also described by Levine et al in 1988, Toxicon 26, 1123-1128. This assay detects okadaic acid with a reported detection sensitivity of 160 pg. However, okadaic acid can be biologically inactivated by a single methyl esterification and therefore, since these procedures give no information on the viable biological activity of a detected toxin, they still require validation by activity-based methods of analysis.
Physico-chemical techniques have also been successfully employed to analyze ng quantities of okadaic acid and dinophysistoxin-1, either indirectly by pre-column derivatisation with 9-anthryldiazomethane (ADAM) followed by liquid chromatography (LC) or directly by LC-linked ion-spray mass spectrometry.
It was recently found that okadaic acid is a potent and specific inhibitor of protein phosphatase-1 (PP1) and protein phosphatese-2A (PP2A), two of four major protein phosphateses in the cytosol of mammalian cells that dephosphorylate serine and threonine residues. These finding were documented by Bialojan et al. in 1988, Biochemical Journal 256, 283-290; Haystead et al. in 1989, Nature 337, 78-81 and Cohen et al. in 1989, J. Biol. Chem. 264, 21435-21438. However, when crude marine extracts from shellfish or phytoplankton are assayed for okadaic acid using a protein phosphatese, it is difficult to evaluate whether the inhibitory response results from heterogenous or homogenous activity. Hence, since okadaic acid is not the only compound which inhibits the enzymatic activity of protein phosphatases, and this may be especially important in the case of shellfish which are filtering organisms, it is virtually impossible to determine the organisms, presence of okadaic acid simply by analyzing the inhibitory effect of a crude sample on a given protein phosphatase.
Thus, there is still a great need for an efficient marine bioscreen for DSP toxins such as okadaic acid and related derivatives.